Discovery and adaptation of CRISPR-Cas systems for genome editing have allowed us to gain an efficient and yet simple tool for genetic manipulation in various fields of molecular biology and biotechnology. One of the most promising applications is the use of CRISPR-Cas9 endonuclease for gene drive systems as a population control strategy for various insect pests of medical and agricultural importance. Use of CRISPR-Cas9 endonuclease in gene drive applications has shown great promise in the

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... atory, particularly for the control of Anopheles gambiae, the major vector of malaria. However, the performance of such gene drives can be limited by the range of available target sequences and by a propensity of existing endonuclease formulations to generate resistant mutations that hinder the gene drive's efficiency. To expand the flexibility of gene drive systems, computational analysis was performed to identify additional Cas9 orthologs and their specificities that could usefully augment the targeting range of endonuclease-based gene drives. Two alternative variants of CRISPR-Cas endonucleases found in the bacterial species Lactobacillus rhamnosus and Bacteroides fragilis were assessed for their potential to expand the targeting space in the genome Anopheles gambiae. In addition, a computational tool was developed that evaluates neighbouring sequences to the target site to measure both its likely functional constraint and its likely propensity for DNA repair that could generate in-frame alleles. Using this approach we were able to generate a prioritized list of Anopheles gambiae target sites for gene drive applications that are less likely to be compromised by resistant alleles.